Type I allergies are a significant health problem in industrialised countries. This type of allergy is caused by the formation of IgE antibodies against antigens carried by the air. These IgE antibodies interact with the mast cells and basophiles, liberating biological mediators such as histamine, and causing allergic rhinitis, conjunctivitis and bronchial asthma in about 20% of the population [(1) Miyamoto, T. (1992). Increased prevalence of pollen allergy in Japan. In Advances in Allergology and Clinical Immunology. P. Godard, J. Bousquet, and F. B. Michel, eds. (Cornforth, UK: The Parthenon Publishing Group), pp. 343-347].
Specific immunotherapy (SIT) is an effective treatment for allergic reactions triggered by specific allergens and basically consists in modulating the immune response in the patient by the regular administration, in increasing concentrations, of the proteins which produce the allergy (allergenic extracts). High doses of the injected allergens induce the high synthesis of IL-12 by the antigen-presenting cells, for example the dendritic cells, which preferably promote the development of T cells which are cooperative virgin cells (nTH) toward TH1 or TH0. This allows a deviation from the immune response of the allergic response type related to the TH2 cells toward a TH1/TH0 type response which leads to the production of high levels of IFN-7 [(2) Akdis, C. A. and Blaser, K. (2000). Mechanisms of allergen-specific immunotherapy. Allergy 55, 522-530]. The immune deviation is reinforced by the induction of the tolerance (anenergy or clonal deletion) of TH2 memory cells under the influence of regulating T cells (TR1) which produce the immunosuppressor cytokines IL-10 and TGF-β [(3) Akdis, C. A., Joss, A., Akdis, M., and Blaser, K. (2001). Mechanism of IL-10 induced cell inactivation in allergic inflammation and normal response to allergens. Int. Arch Allergy Immunol. 124; 180-182]. The reduction in the activation and proliferation of the TH2 cells is accompanied by reduced production of IL-4, and of IgEs by the B cells. The reduction in the activity and infiltration of the TH2 cells into the nasal and bronchial mucus results in reduced IL-5 synthesis, allowing a reduction in eosinophile infiltration which leads to a great reduction in the liberation of inflammatory mediators such as MBP and ECP proteins. The new specific clones of T cells of predominant phenotype allergen TH0 produce a mixture of TH1 and TH2 type cytokines promoting the production of a large quantity of specific IgG allergen antibodies by the B cells. On the other hand, the high levels of IL-10 induce the high synthesis of specific IgG4 allergen antibodies. These two types of specific antibodies can act as blocking antibodies providing the intersection of the IgE antibodies combined with their receptors in the mast cells and therefore inhibiting the degranulation and liberation of histamine [(4) Moverate, R. (2003). Immunological mechanisms of specific immunotherapy with pollen vaccines: implications for diagnostics and the development of improved vaccination strategies. Expert Rev. Vacc. 2, 85-97; (5) Wachholz, P. A., Soni, N. K., Till, S., and Durham, S. R. (2003). Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J. Allergy Clin. Immunol. 112; 915-922]. They also block the collection of IgE-mediated antigen by the antigen-containing cells, and this suppresses the immune reaction to the allergens.
The allergen extracts isolated from natural sources are complex mixtures of proteins and other molecules. Their composition, and therefore allergenicity, depends on the material used, which varies according to the ambient conditions, the state of maturation in the case of pollens and the conditions of growth of mites, etc., in the case of fungi, etc. Some extracts can even contain an inadequate concentration of major allergens and can even be contaminated with undesirable components to which the patient is not allergic, or both. Current immunotherapy uses complete allergen extracts exclusively, and this leads to a number of problems such as: serious adverse reactions owing to the reactivity of the vaccine with IgE of the effector cells, appearance of new sensitisation to other allergens present in the vaccine after immunotherapy, and difficulties in standardisation of the allergen extracts.
All this means that immunotherapy is not a treatment which is as safe and effective as desired. Better knowledge of the pathogenesis of allergy and of the mechanisms of the specific immunotherapy has made it possible to get closer to the solution to the aforementioned problems. Knowledge of the influence of the IgE-mediated antigen in the specific allergen response TH2 has increased efforts to create allergens which do not bind IgE. The main object of current specific immunotherapy is to modify the allergen with the aim of inactivating the IgE epitopes, thus reducing and even eliminating the bond to IgE and consequently the adverse reactions [(6) Valenta, R. and Linhart, B. (2005). Molecular design of allergy vaccines. Curr. Opin. Immunol. 17, 1-10]. In this way, the modified allergen will be directed toward the T cells by a phagocytosis/pinocytosis-mediated antigen collection mechanism, preventing the IgE intersection and the presentation of IgE-dependent antigen. This induces a balance in TH0 or TH1 cytokine production by the T cells, less production of IgE and more production of IgG by the B cells; all this will lead to the induction of TH2 type T cell tolerance without a risk of anaphylaxis. Progress in recombinant methods of obtaining allergens and allergen derivatives has allowed a great increase in the capacity for developing new vaccines for the treatment of allergies. This has been possible owing to the possibility of mutating or deleting significant amino acids of IgE epitopes, as well as the fractionation and oligomerisation thereof for obtaining hypoallergenic vaccines. These molecules which have a lesser capacity to bind IgE but maintain their reactivity toward T cells may be administered in greater doses, allowing faster and safer immunotherapy with a smaller number of injections. In addition, recombinant allergens may be produced on a large scale in fermentation tanks, using microbial expression systems, and the purification thereof is more efficient and inexpensive than that of their natural equivalents. The use of hypoallergenic derivatives in immunotherapy has previously been described using fragments of trimers of Bet v 1 [(7) Niederberger, V., Horak, F., Vrtala, S., Spitzauer, S., Krauth, M. T., Valent, P., Reisinger, J., Pelzmann, M., Hayek, B., Kronqvist, M., Gafvelin, G., Grönlund, H., Purohit, A., Suck, R., Fiebig, H., Cromwell, O., Pauli, G., van Hage-Hamsten, M., and Valenta, R. (2004). Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc. Natl. Acad. Sci. U.S.A. 101, 14677-14682], multi-allergenic hybrids of bee venon proteins (Api m 1, 2, 3) [(8) Schmid-Grendelmeier, P., Karamloo, F., Müller, U., Housley-Marcovic, Z., Soldatova, L., Zumkehr, J., Kemeny, D. M., Kündig, T., Reimers, A., von Beust, B. R., Salagianni, M., Akdis, M., Kussebi, F., Spangfort, M. D., Blaser, K., and Akdis, C. A. (2005). Prevention of allergy by a recombinant multi-allergen vaccine with reduced IgE binding and preserved T cell epitopes. Eur. J. Immunol. 35, 3268-3276] and mutated fusions of Par j 2 and Par j 1 for eliminating their tertiary structure.
Some authors mention that allergenic vaccines should not be made with hypoallergens since the bond to IgE could facilitate the capture and presentation of the allergen by the professional antigen-presenting cells, as dendritic cells and activated B lymphocyte cells, which express surface receptors for IgE of both high affinity and low affinity. This approach has a special meaning when using the sublingual route in immunotherapy. The intersection of high affinity receptors can also lead to a reduction in the response of the T cells toward the allergens [(9) Allam, J. P., Novak, N., Fuchs, C., Asen, S., Berge, S., Appel, T., et al. (2003) Characterization of dendritic cells from human oral mucosa: a new Langerhans' cell type with high constitutive FcεRI expression. J. Allergy Clin. Immunol. 112, 141-8. (10) von Bubnoff, D., Matz, H., Frahnert, C., Rao, M. L., Hanau, D., de la Salle, H., Bieber, T. (2003) FIεRI induces the triptophan degradation pathway involved in regulating T cell responses. J. Immunol. 169, 1810-1816]. Many researchers are still making this type of vaccine without introducing deletions [(11) Batard, T., Didierlaurent, A., Chabre, H., Mothes, N., Bussieres, L., Bohle, B., et al. (2005) Characterization of wild-type recombinant Bet v la as a candidate vaccine against birch pollen allergy. Int. Arch. Allergy Immunol. 136, 239-249. (12) Jutel, M., Jaeger, L., Suck, R., Meyer, H., Fiebig, H., Cromwell, 0. (2005) Allergen-specific immunotherapy with recombinant grass pollen allergens. J. Allergy Clin. Immunol. 116, 608-13. (13) Niederberger, V., Horak, F., Vrtala, S., Spitzauer, S., Krauth, M. T., Valent, P., et al. (2004) Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc. Natl. Acad. Sci. U.S.A. 101, 14677-82. (14) Cromwell, O., Fiebig, H., Suck, R., Kahlert, H., Nandy, A., Kettner, J., et al. (2006) Strategies for recombinant allergen vaccines and fruitful results from first clinical studies. Immunol. Allergy Clin. N. Am. 26, 261-81].
Parietaria is a genus of dicotyledon weed from the Urticaceae family, and the Urticales order. Various species of the Parietaria genus are widely and abundantly distributed along the Mediterranean coast [(15) Colombo, P., Duro, G., Costa, M. A., Izzo, V., Mirisola, M., Locorotondo, G., Cocchiara, R., and Geraci, D. (1998). An update on allergens. Parietaria pollen allergens. Allergy 53, 917-921]. The most common species are P. judaica and P. officinalis, but other species such as P. lusitanica, P. mauritanica, and P. cretica, may have some presence in some regions. Nevertheless, the Mediterranean regions are not the only ones where Parietaria pollen can be found, since its presence has been described in the South of England, Austria, temperate regions of central and eastern Europe Australia and California [(16) Colombo, P., Bonura, A., Costa, M., Izzo, V., Passantino, R., Licorotondo, G., Amoroso, S., and Gerasi, D. (2003). The allergens of Parietaria. Int. Arch. Allergy Immunol. 130, 173-179; (17) Carreira, J. and Polo, F. (1995). The allergens of Olea europaea and Parietaria spp. and their relevance in the Mediterranean Area. Allergy Clin. Immunol. News 7, 79-84]. A significant characteristic of Parietaria is the long pollenisation period which lasts for a plurality of months and results in the presence of almost perennial symptoms in patients who are allergic to Parietaria, ranging from slight rhinoconjunctivitis to severe asthma. It should be noted that the normal light monospecific sensitisation to Parietaria involves the sensitisation to various species of this genus, since significant crosswise reactivity has been demonstrated between differing species of Parietaria. 
Various papers have been presented on the purification and characterisation of allergenic fractions of the two most common species which are P. judaica and P. officinalis. These fractions have molecular weights in the range from 10-14 kDa and are responsible for virtually the entire allergenic power of their extracts [(16) Colombo, P., Bonura, A., Costa, M., Izzo, V., Passantino, R., Licorotondo, G., Amoroso, S., and Gerasi, D. (2003). The allergens of Parietaria. Int. Arch. Allergy Immunol. 130, 173-179; (18) Ayuso, R., Carreira, J., Lombardero, M., Duffort, O., Peris, A., Basomba, A., and Polo, F. (1993). Isolation by mAb based affinity chromatography of two Par j isoallergens. Comparison of their physicochemical, immunochemical and allergenic properties. Mol. Immunol. 30, 1347-1354; (19) Polo, F., Ayuso, R., and Carreira, J. (1990). HPLC purification of the main allergen of Parietaria judaica pollen. Mol. Immunol. 27, 151-157; (20) Polo, F., Ayuso, R., and Carreira, J. (1991). Studies on the relationship between structure and IgE-binding ability of Parietaria judaica allergen I. Mol. Immunol. 28, 169-175]. The development of recombinant DNA technology has enabled the molecular characterisation of Parietaria pollen allergens to be completed: the two major allergens of P. judaica pollen known as Par j 1 and Par j 2 have been cloned and sequenced [(21) Duro, G., Colombo, P., Costa, M. A., Izzo, V., Porcasi, R., DiFiore, R., Locorotondo, G., Mirisola, M. G., Cocchiara, R., and Geraci, D. (1996). cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen. FEBS Lett. 399, 295-298; (22) Costa, M. A., Colombo, P., Izzo, V., Kennedy, H., Venturella, S., Cocchiara, R., Mistrello, G., Falagiani, P., and Geraci, D. (1994). cDNA cloning expression and primary structure of Par j I, a major allergen of Parietaria judaica pollen. FEBS Lett. 341, 182-186; (23) Amoresano, A., Pucci, P., Duro, G., Colombo, P., Costa, M. A., Izzo, V., Lambda, D., and Geraci, D. (2003). Assignment of disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen. Biol. Chem. 384, 1165-1172]. Both allergens belong to the family of non-specific lipid transfer proteins (ns-LTP) and possess a signal peptide in their terminal region which, after processing, gives rise to proteins having a molecular weight of 14,726 and 11,344 Da respectively and having about 45% of identical residues. The possible IgE-binding linear epitopes in both allergens, which would be situated in structurally related zones, have been described [(24) Asturias, J. A., Gómez-Bayón, N., Eseverri, J. L., and Martínez, A. (2003). Par j 1 and Par j 2, the major allergens from Parietaria judaica pollen, have similar immunoglobulin E epitopes. Clinical and Experimental Allergy 33, 518-524]. These regions are the targets which will be acted upon in order to be able to obtain the optimum hypoallergenic molecules for the treatment of the allergy to P. judaica pollen.
The ns-LTP are well known for their capacity to complete in vitro the intermembrane interchange and/or the transfer of polar lipids [(25) van Ree, R. (2002). Clinical importance of non-specific lipid transfer proteins as food allergens. Biochem. Soc. Trans 30, 910-913]. Two main families have been characterised in plants, LTP1 with a molecular mass of approximately 9 kDa and LTP2 with a molecular mass of approximately 7 kDa. Allergens belonging to the LTP family have been identified in plants other than foods, where they have been widely studied. Thus, Hev b 12 from the latex of Hevea brasiliensis is a basic 9.3 kDa protein which demonstrates about 65% sequence identity with allergenic LTPs of fruits of the Rosaceae family [(26) Beezhold, D. H., Hickey, V. L., Kostyal, D. A., and et al. (2003). Lipid transfer protein from Hevea brasiliensis (Hev b 12), a cross-reactive latex protein. Ann Allergy Asthma Immunol 439-445]. In addition, some pollen allergens have been described as LTPs, such as Art v 3 of Artemisia vulgaris [(27) Díaz-Perales, A., Lombardero, M., Sanchez-Monge, R., and et al. (2000). Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE-binding capacities. Clin Exp Allergy 1403-1410] and Ole e 7 of Olea europaea [(28) Tejera, M. L., Villalba, M., Batanero, E., and Rodriguez, R. (1999). Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J. Allergy Clin. Immunol. 797-802; (29) Rodriguez, R., Villalba, M., Batanero, E., and et al. (2002). Allergenic diversity of the olive pollen. Allergy 6-16] which are of 9-10 kDa and have 30-55% sequence identity with allergenic LTPs of foods. The major allergens of P. judaica, Par j 1 and Par j 2, which have about 45% sequence identity with one another, on the other hand, have higher molecular weights than normal in the LTPs family; 14.7 and 11.3 kDa, respectively [(16) Colombo, P., Bonura, A., Costa, M., Izzo, V., Passantino, R., Licorotondo, G., Amoroso, S., and Gerasi, D. (2003). The allergens of Parietaria. Int. Arch. Allergy Immunol. 130, 173-179]. However, although both allergens have a similar structure to that of the LTPs, they have moderate levels of identity with food LTPs in the common sequence region (28% between Par j 1 and peach LTP).
WO2005/085278 discloses the construction of a fusion protein comprising the two major allergens of Parietaria judaica, wherein the three-dimensional structure of the fusion protein has been disrupted by replacement of certain cysteine residues in the primary sequence of each allergen (more specifically the cysteine residues involved in the formation of disulphide bridges) so that the sequences of allergens maintain essentially the same length. According to later experiments, this should lead to a protein that is 1000 times less allergenic than natural allergens.
The inventors of the present invention have discovered that a surprisingly large reduction in allergenicity can be obtained not only by disrupting the three-dimensional structure of the allergen but also by deletion of some IgE-binding sites (known as B epitopes) and that, most surprisingly, this does not lead to a reduction in immunogenicity.
The present invention discloses for the first time differing chimeric proteins obtained by binding fragments of the two allergens of Parietaria judaica (Par j 1 and Par j 2) containing a smaller number of IgE-binding epitopes, as well as differing methods and intermediates for obtaining them. Not only is the three-dimensional structure of the chimeric proteins of the present invention disrupted but also certain B epitopes have been deleted. The chimeric proteins according to the present invention can be called hypoallergenic with an allergenicity reduced by 99.99%, as they have a lower capacity to bind IgE antibodies based on: i) in vitro ELISA, ELISA inhibition and immunodetection tests using mixtures of sera from patients allergic to P. judaica; ii) in vivo tests of cutaneous reactivity in patients allergic to P. judaica; and iii) in vitro EAST inhibition test with individualised sera from patients allergic to P. judaica. The chimeric proteins according to the present invention, on the other hand, maintain their immunogenic capacity, as demonstrated by lymphoproliferation tests on peripheral blood mononuclear cells (CMSP) from 13 patients allergic to P. judaica. 
The allergenic extracts are complex mixtures of proteins and non-protein molecules. The increasing use of techniques for detecting the levels of specific IgE against the components of an extract has made it possible to demonstrate that allergic patients usually have reactivity toward various components. Cases of allergic patients who react only to a single allergen are rare. Since allergenic extracts have obvious problems in immunotherapy, one solution is to group as many therapeutic properties as possible in a single molecule.